Implementing gene-based assays on a robotized genosensor for environmental surveillance in offshore marine operations

This project is addressing next generation monitoring technology to meet today?s and future regulatory demands as well as society?s acceptance to operate offshore in an environmentally-friendly manner. It includes an important level of novelty for the offshore industries, environmental managers and the scientific world. The use of DNA-based technology applied to the marine environment is increasing and can allow for quick on-site determination of environmental status. Here, we will use the Environmental Sample Processor (ESP), a real-time unmanned platform enabling cutting-edge DNA-based measurements at sea to detect subtle changes in bacterial community used as signatures of environmental pollution by oil. Crude oil pollution affects rapidly the bacterial community composition with the resulting dominance of some few oil-degrading bacterial species. The changes in bacterial community can be used as an effective biosensor for oil contamination in the marine environment and the ESP is a robotized platform enabling their real-time monitoring at sea. Previously, a selection of oil-specific bacteria was made and DNA-based protocols for ESP tested to reveal and quantify these bacteria using PCR. Here the project explores new nucleic acids-assays for use on ESP, particularly those related to function for oil degradation, and new analytical capabilities of the next-generation of ESP such as surface plasmon resonance (SPR). The outcome of WP1 and WP2 was to identify microbial genes involved in biodegradation of oil for qPCR primer design. A metatranscriptomics pipeline was used to identify genes that are activated in response to oil. From this analysis, and database/literature search, alkane monooxygenase gene marker was selected for detection of alkane degradation. Alkan 1 mono-oxygenase (AlkB) is an enzyme found in many types of bacteria, and is involved in the bacterial degradation of alkanes (an important component of oil) in nature. The corresponding degenerate primer (alkB-deg) was able to target conserved regions of various alkane degrading bacteria including marine bacterium SAR92 (which has been found to be significantly upregulated in the presence of oil), and other oil-dependent bacteria such as Alcanivorax, Oleiphilus and Oleispira, all well-known oil degraders. For aromatic hydrocarbons, two new markers were selected: 3,4-dihydroxyphenylacetate 2,3-dioxygenase and aromatic ring-hydroxylating dioxygenase. The corresponding designed degenerate primers (hpaD-deg and nahAc-deg, respectively) target regions among phylogenetically different bacteria families such as Colwelliaceae, Oceanospirillaceae (hpaD-deg) and Piscirickettsiaceae (Cycloclasticus) and Sphingomonadaceae (nahAc-deg). These primers were tested on water samples collected for two seasons (winter and summer) in mesocosm experiments (see report 2018). The degenerate primers did not amplify the intended targets as well as expected and did not reflect well enough the increase in major microbial player abundance. Quantifying Oleispira (Oceanospirillaceae ) served as equally good or better signal of oil exposure as alkB gene counts, particularly in the summer and for the light crude oil. The ESP is underway to be a new mobile underwater device, with new analytical modules such as surface plasmon resonance (SPR). WP3 is addressing this technology transition and the results after a phase of development with a morpholino probe for Oleispira antarctica designed during the previous Petromaks2 MOAB project are promising. The SPR module has been tested under a series of analyses to address challenges related to the envisioned 16S rRNA capture approach using magnetic-beads and included also testing Environmental Sample Processor (ESP) compatible procedures. A master student from the University of Stavanger was involved in the analyses. After several failed attempts, a viable Oleispira antarctica culture has been established in the laboratory and was used for harvesting RNA for capture and SPR experiments. Our results demonstrated the ESP compatible chemistry is suitable for the morpholino-probe based detection of short oligonucleotides on the SPR instrument but technical challenges remain to use actual Oleispira RNA for SPR detection. After a bench test and optimization of the assay for use on the ESP at NORCE, DTU-Denmark has just completed the analysis of a selection of field-relevant seawater samples with the ESP technology in the laboratory. Assays using Colwellia, Oleispira and Cycloclasticus taxonomic gene (developed in #215598), and both functional gene AlkB and nahAc-deg were used as targets. The data are under analysis. Overall the results show a rather good response of the taxonomic assays to level of pollution but the functional assays response to pollution are more variable.

Det er viktig å sikre at petroleumsnæringen driver deres virksomhet på en miljømessig forsvarlig måte. Prosjektets viktigste leveranse har vært å demonstrere at en endring av olje-relatert mikrober kan fungere som et alternativ til å påvise olje lekkasje eller søle i vann, og at ESP-roboten faktisk kan gjøre dette i sanntid i felt. Dette gjelder særlig til mindre lekkasje der eksisterende teknologier feiler å påvise olje. Dessuten, man måler en faktisk konsekvens og mulig risiko for miljø når naturlige mikrobielle forekomster er endret forårsaket av olje i vann. Petroleumsnæring mangler system for å oppdage mindre akutt forurensning eller lekkasjer fra undervannsinnretninger på norsk sokkel, slik at raskt avbøtende tiltak for å minske risiko tas. PTIL har nylig gjennomført en tilsynskampanje r og det har ført til mye arbeid i næringen, og en oppdatering av NOROGs sin retningslinje 100. Erfaringen og teknologien brukte i dette prosjektet kan også være nyttig for denne prosessen videre.

Monitoring technologies are demanded for today's and future regulatory demands and society's acceptance to operate with care for the environment, particularly in sensitive marine areas. Novel solutions with devices capable of operating autonomously and in near-real time at sea like the Environmental Sampling Processor (ESP) exist. Here, this fully autonomous genosensor will be adapted. Protocols mimicking ESP operation will be used towards the detection of specific microbial genes involved in oil biodegradation as signatures of accidental oil occurrence at sea. To that goal, several research activities will be performed in the laboratory first then validated with ESP in situ. First identification of bacteria involved in degradation of particular oil fractions will be carried out. Then, information about highly abundant functional genes encoding enzymes involved in that degradation will be gained. Finally molecular assays targeting these genes will be designed (WP1).In WP2, the influence of environmental variables (presence of organic matter, seasons) and different oil types on bacterial gene assay performance and shift under exposure with oil will be evaluated to insure their robustness. As ESP trajectory development is progressing toward a third generation (3G-ESP) with new analytical modules like surface plasmon resonance (SPR), transfer of the gene assays on SPR will be conducted with adapted protocols for that module. Finally, WP 4 will demonstrate the full operability (from detection to communication) of the ESP deployed or used for Norwegian water to perform gene-based assays in situ. Proving that will be a significant step forward for the acceptance of this device as a tool for in situ surveillance by operators. This project will place Norwegian operators in a leading position regarding future environmental technologies and monitoring practices in the Norwegian Continental Shelf and will contribute significantly to their license to operate goal.